Rapid microbial detection

ABSTRACT

Devices and methods are provided to detect the presence of bacteria and small microorganisms, and to identify various microbial attributes rapidly.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/893,689 filed Nov. 24, 2015, titled “Rapid Microbial Detection,”which is a national stage application of International PatentApplication No. PCT/US2014/000137 filed May 30, 2014, titled “RapidMicrobial Detection,” which claims priority to U.S. provisional patentapplication Ser. No. 61/956,065, filed May 31, 2013, titled “RapidMicrobial Detection,” the entirety of all of which is incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates to devices and methods to detect thepresence of microorganisms, such as bacteria, and to identify variousmicrobial attributes rapidly.

BACKGROUND

Each year more than 2 million children around the world under the age offive die from bacterial pneumonia alone. In addition, foodborne andwaterborne diseases (normally involving bacteria) kill about 2.2 millionpeople globally, generally in regions where analytical devices areunavailable. Thus, microorganism detection technologies that areportable and deployable would provide tremendous health benefitsworldwide.

Current methods to detect microorganisms normally require more than oneday to provide results. The traditional culture-based method takes evenlonger (1-7 days) and is frequently used on account of its low cost andgreater sensitivity. As a result, various treatment decisions involvingpatients are delayed until availability of results. In locations withoutaccess to a well-equipped laboratory that can perform sample culturing,the sample has to be shipped, thereby adding delay, cost, and logisticissues.

Because of the delays and costs involved, treatment decisions areinitially based on “educated guesses” followed by a more specificdecision based on the laboratory results or empirical results with aparticular medication. For instance, a physician may prescribe abroad-spectrum antibiotic to cover a wide variety of bacteria withoutknowing if the infection is bacterial. If the presence of bacteria isconfirmed and if the identity of the bacteria is determined, then theantibiotic is changed to one that is more appropriate for thosebacteria. A patient's response is also a factor in selecting an optimaltreatment.

More rapid analyses are critical in the treatment of bacterialinfections since morbidity and mortality have been directly correlatedto the early initiation of appropriate antibiotic therapy.

Similarly, in the case of food manufacturing, food may be shippedwithout waiting for the results due to their perishable nature. If thepresence of bacteria is confirmed in the sample, then the food isrecalled. At times, a customer may have consumed the food before theseresults are available. This is one of the major causes for various foodcontamination outbreaks. In the U.S. alone, it is estimated that thereare 9.4 million episodes of foodborne illnesses resulting in 1,300deaths per year and direct healthcare costs of treating people ofapproximately $152 billion a year. And companies lose hundreds ofmillions of dollars due to costs of recall, rejected batches,compensation, market share, and loss of reputation. Additionally, due toglobalization and sourcing of raw materials from all over the world, araw material may be used in a process before ascertaining the presenceof bacteria. If harmful bacteria are found to be present in the rawmaterial, this may result in the rejection of a processed batch causingsubstantial financial harm to the manufacturer. Thus, rapid and portabledetection technologies would provide tremendous health as well aseconomic benefits worldwide.

Most of the current rapid methods rely on bacterial identification basedon antibody recognition or specific nucleic acid sequence binding. Whilethese methods offer an increase in speed (compared to traditionalculture) and specificity of detection, they are more expensive comparedto the traditional culture methods and may not be as accurate. Further,while in principle almost all species can be identified on the basis oftheir specific nucleic acid sequence, practical limits on multiplexingmean that these techniques can identify only a few microorganisms at atime. As a result they are not an effective screening technique wherethe pathogen may be one of a large number of possible pathogens. Othertechniques such as mass spectrometry based identification may offerlower cost per test but require highly skilled operators and veryexpensive equipment in a specially equipped laboratory. As a result,many small to mid-sized organizations and businesses, includinghospitals and agricultural processing plants, tend to use traditionalculture method despite the longer time to diagnosis.

Clearly, there is a need for methods and devices that can be used toidentify microorganisms at the point of need (for example, a patient'sbedside, manufacturing site, farm, etc.) and that provide rapid andaccurate results at a relatively low cost.

SUMMARY

The present invention provides a rapid and integrated detection andidentification system for various bacteria and other unicellular andmulticellular microorganisms. It requires relatively small amounts ofsample and processes samples and identifies microorganisms withoutculturing and without manual intervention steps typically found indevices used in the field. The detection and identification systemoperates rapidly compared to previously known devices, providing resultsin about one hour or less. Portable devices embodying the detection andidentification system are contemplated for use in the field in a widevariety of locations and environments. As used herein, the term“bacteria” is used for convenience rather than reciting each time“bacteria and/or other small microorganisms” unless it is clear from thecontext that only bacteria or some other microorganism is beingdiscussed.

Devices and related methods according to the present invention generallycomprise a microfluidic separation stage or component and an infrared(IR) spectrometric stage. An identification stage preferably is alsopart of the detection system. Preferably, the identification stagefurther includes both a spectral analysis stage and a result reportingstage. The identification of microorganisms in the sample is performedat the point-of-use or remotely, and the data generated for theidentification of microorganisms in the sample may be stored andanalyzed locally or output for remote analysis or storage.

A preferred embodiment of the detection systems and devices according tothe present invention is applicable to the analysis of bacteria inbodily fluids such as blood, and is configured to lyse blood cells inblood samples and remove detritus and other debris that might interferewith detection and identification. Components of the system generallyprovide the capability to preferentially select and to concentrate thecells of interest (for example, bacteria, fungi, yeast, which may bespherical, non-spherical, or deformable). Thus, in other embodiments,the detection system identifies microorganisms such as fungi, and smallunicellular and multicellular organisms.

The detection system processes and detects and identifies microorganismsin liquid samples such as whole blood, plasma, serum, saliva, urine,cerebrospinal fluid, water, and fruit and vegetable juices. The volumeof such samples is relatively low compared to previously known devices,and preferably ranges from about 0.1 mL to about 10 mL. The ability toprocess larger sample volumes of about 1-10 mL aids in the detection andidentification by increasing the probability of the microorganismcontaminant in the sample and its amount. Most current diagnosticsystems utilize lower volumes of sample (less than about 0.5 mL). Thedetection system also processes other types of samples such as meat,produce, processed food, dairy products, poultry products,pharmaceutical process streams, bulk drug substance, and final drugproduct.

The microfluidic separation stage preferentially selects intactmicroorganisms. It also selectively lyses non-microbial cells. Notably,the microorganisms in the sample do not need to be cultured or otherwiseisolated from the sample prior to application of the sample to thedetection system prior to analysis.

The microfluidic separation stage optimally contains a measurementpreparation stage to concentrate the intact bacteria and then to director channel them to an IR-compatible surface. Optimally, themicroorganisms in the sample to be analyzed are concentrated tofacilitate detection and analysis by reducing the sample's volume or byfiltration. And, notably, the detection system according to the presentinvention separates out and removes the debris from the lysis ofnon-microbial cells without using nucleotides (such as DNA), antibodies,or other ligands and reagents that specifically recognize and bind themicroorganisms of interest, such as bacteria.

In another preferred embodiment, the infrared spectrometric stageutilizes beam condensers to reduce the width of the IR beam to which themicroorganisms in the sample are exposed. Preferably, the IRspectrometric stage also contains a single-point or a focal plan arraydetector.

Preferably, a measurement stage is provided to measure the IR signaturesof the microorganisms in transmission mode. In another of its aspects,the measurement is performed using other modes commonly used in infraredspectroscopy such as attenuated total reflection (ATR), and diffusereflection infrared Fourier transform (DRIFT).

In preferred embodiments, the measurement stage includes ultrathinpolycrystalline silicon filter. In other embodiments, the measurementstage may include a flow cell or detection window.

Other embodiments of the present invention also include a wastecollection stage. Preferably, a computer system or microprocessor areincluded in the detection system to collect, digitize and process theacquired data. Embodiments including a reporting stage to provide outputof analyzed data are preferred. Preferably, the detection system alsoincludes a control stage.

The identification stage of the present invention preferably utilizesstandard chemometric approaches to compare sample IR spectra againstthose in a reference database in order to identify the genus, species,or strain of bacteria and other microorganisms detected and analyzed. Inspecific use situations, reference spectra in the database would includeIR spectra of particular predetermined microorganisms whose detectionand identification are desired in an emergency or acute setting. It iscontemplated that a neural network or other computational approaches maybe used to identify the bacteria and other microorganisms in a sample.

In yet another aspect of the present invention, a microfluidicseparation device is provided. The device preferably includes a firstseparation stage to separate debris and smaller analytes andcontaminants from intact microbial cells, a lysis stage to selectivelylyse non-microbial cells, and a second separation stage to separatelysed cells and cellular debris from the microbial. The first separationstage is particularly useful for the analysis of microorganisms presentin a blood sample. If the sample matrix is cerebrospinal fluid or anenvironmental sample, such as pond water for example, then the firstseparation stage may be unnecessary as would readily be determined by aperson skilled in the art. The second separation stage optionally mayoperate by filtration or other methods known to persons skilled in theart.

Another aspect of the present invention relates to a method of detectingor identifying microorganisms in a sample by introducing the sample forprocessing into a detection system as described above, processing thesample and then obtaining the analyzed data output from the detectionsystem. Contemplated microorganisms to be detected and/or analyzed insuch methods include prokaryotes, eukaryotes, protozoans, filamentousfungi, and algae. The detection system is also intended to distinguishdifferent species and strains of a specific genus, for instancedifferent bacterial species from the genera Staphylococcus, Escherichia,Listeria, Salmonella, Streptococcus, Klebsiella and Campylobacter.

DESCRIPTION OF THE DRAWINGS

The following figures, which are described below and which areincorporated in and constitute a part of the specification, illustrateexemplary embodiments according to the disclosure and are not to beconsidered limiting of the scope of the invention, for the invention mayadmit to other equally effective embodiments. The figures are notnecessarily to scale, and certain features and certain views of thefigures may be shown exaggerated in scale or in schematic in theinterest of clarity and conciseness.

FIG. 1 is a schematic showing the major components of the integrateddetection and identification system and their functions.

FIG. 2 is an example of a plot of infrared absorbance versus wavelengthfor a particular species of bacteria.

FIG. 3 is a schematic showing another embodiment of the integrateddetection and identification system.

DETAILED DESCRIPTION

There are several highly important features desired in rapid bacterialdiagnostics—speed to result, the ability to identify a broad range ofpathogens accurately, and simple/inexpensive operation. Historically,diagnosis of bacteria has relied on growth-based technologies in orderto expand the population of bacteria to detectable levels. Most of therapid diagnostic approaches used to-date have focused on reducing thetime-to-result (TTR) post-culturing and/or through the use ofproprietary media, which accelerate the growth of selected bacterialspecies. The reductions in TTR therefore have largely been incrementalsince the time needed for the bacteria to grow is the rate-limitingstep.

Infrared and Raman spectroscopy have been widely studied and used fordetection of various analytes, including bacteria. The spectroscopyrelies on the unique patterns generated by the vibrational energies ofthe bonds that exist between atoms (for example, C—O, C—H, etc.). Helmet al. [1]

In the above studies, the identification of bacteria using Fouriertransform infrared spectroscopy (FTIR) has been performed on culturedand isolated bacteria. Additionally, large quantities of bacteria(50-100 micrograms) are utilized in the analysis, which also usescomplex procedures for instance, drying of bacteria on the surface of areflective element to form a Biofilm. The analysis is performed inwell-equipped laboratories involving highly skilled analysts and takesup to one week. El-Sayed et al. [2] report a method for analyzing threecommon bacteria in otitis media while submerged in a biologic fluid. Thethree most common bacteria studied were Streptococcus penumoniae,Haemophilus influenza, and Morazella catarrhalis.

In general, the present invention is useful in various applications andlocations where rapidly determining the presence and identity ofbacteria and other very small unicellular and multicellular organisms isof importance. These include, for example, medicine (to determinebacterial infections), food safety, pharmaceutical quality control(including biotechnology products), environmental monitoring, andbiosurveillance. The invention thus enables, for example, detection andidentification of bacteria rapidly, at low cost, and at the point ofneed permitting appropriate actions to be taken. These may include, forinstance, rejecting a batch of food or pharmaceutical, subjecting thebatch to additional treatment, or determining the course of treatmentfor a patient. The invention also relates to methods to detect thepresence of bacteria and other very small unicellular and multicellularorganisms, and to identify different attributes, for example, ofbacteria, such as the species and strain, in any sample matrix using anintegrated device.

In one preferred embodiment, the present invention offers the ability touse a portable and integrated device to detect all bacteria from anysample matrix (as long as the matrix is a liquid, suspension, gel,slurry, or is liquefied such as a solid that has been digested usingenzymes or put through a blender with some liquid) without the need forculturing, using tagging or binding reagents such as antibodies ornucleic acid primers, requiring analysis at well-equipped laboratories,or requiring the services of highly skilled technicians.

The microfluidic separation component according to the invention iscapable of separating intact bacteria (or other microorganisms) from thesample matrix, without a need for organism-specific reagents such asantibodies or nucleic acid probes. Preferred sample matrices are fluidssuch as water, whole blood, milk and juice. Other appropriate samplesinclude plasma, serum, saliva, urine, cerebrospinal fluid, food (such asmeat, produce, processed food, dairy products, and poultry products),pharmaceutical process streams, bulk drug substance, and final drugproduct.

The detection component according to the invention preferably is aninfrared spectrometer that is capable of measuring the IR spectrum ofintact bacteria (or other small organisms), for example, in amicrochannel or filter.

For instance, in clinical use the diagnosis is achieved by placing adisposable microfluidic chip (FIG. 3 ) in the appropriate slot of adevice according to the present invention, inserting a tube containingthe clinical sample in the sample port, and initiating the analysis bypressing a start button. The identity of the bacteria will be displayedon an LCD screen on the instrument.

The results can also be communicated and stored in any of severaldigital and hard copy formats. The clinical sample (for example, blood)is prepared for infrared measurement by first separating blood cellsfrom intact bacteria, then selectively lysing the non-bacterial cells,then separating the lysis debris from intact bacteria, and finallyconcentrating the intact bacteria on a small IR-compatible surface. Theinfrared spectra of the bacteria on the surface is measured using aspectrometer, which may be aided by beam condensers, objective lenses,and apertures. These spectra are then compared against those in areference database to enable identification.

The invention relates, in part, to the discovery that bacteria in asample can be concentrated sufficiently, for reliable identification byinfrared spectroscopy, without culturing and that the infrared spectrumof a bacteria provides a unique fingerprint of the bacteria that aids inspecifically identifying it. Furthermore, the embodiments described inthis specification substantially minimize the need for handling andprocessing a sample compared with the purification and concentrationtechniques in conventional detection systems.

The above benefits arise from the unique sequence of unit operations andtheir integrations as described in this application. For instance, useof mechanical forces to lyse non-bacterial cells by itself would notlend itself towards simple sample preparation. However, by combining itwith a separation step before and after, it enables simple andeffectively reagentless sample preparation. Also, most methods focus onlysing bacteria in order access their DNA or other bacterial biomarkers.The operational steps therefore not only have to clean up non-bacterialcomponents but also separate the targeted biomarker from other bacterialcomponents.

By focusing on isolating intact bacteria, not only is the operationsimplified, but also the intact bacteria can be concentrated to levelssuitable for detection and identification easily without requiringculturing. While the microfluidic chip containing concentrated intactbacteria could be used with various measurements (for example, colonycultivation, mass spectrometry, PCR, etc.), the preferred embodimentuses a technique such as infrared spectroscopy. This ensures measurementin a few minutes, enables label-free identification, and provides theability to identify multiple pathogens from a single measurement.

The preferred detection device is portable for use in the field.Identification routinely can be made in well less than one hour becausethe sample does not require culturing and related manual interventionsteps, which is required for conventional identification devices. Forexample, where a conventional device might require 1-5 days to culturethe sample, the system of the present invention might require only asmuch time as to process the sample volume using the microfluidiccomponent. Similarly, the time to generate a result, for example, anidentification of the bacterial component will generally be on the orderof minutes rather than tens of minutes or of hours.

Preferred detection devices also include an identification module thatprovides reference spectra for the bacteria in a sample and can generatea local identification. Alternatively, IR spectral information may beprocessed at a central location and reference spectra may be utilizedthrough the internet, telephone, radio or other data transmissiontechnologies. The detection is expected to be capable of detecting about10 colony forming units/mL or better. The false positive rates areexpected to be less than about 5% with the accuracy of identificationbeing greater about 90%, 95%, 98% and 99% or better. These falsepositive and accuracy rates are comparable to those of conventionaldevices without the effort, training, and time needed to generate aresult using these devices. Examples of bacteria that can be detectedusing this device include members of the Staphylococcus, Escherichia,Listeria, Salmonella, Streptococcus, and Campylobacter genera.

In one embodiment, the invention provides an integrated device fordetecting bacteria in various matrices, where the device comprises: (a)a microfluidic separation stage to separate bacteria from the rest ofthe sample matrix, thereby enriching their concentrations, (b) ameasurement preparation stage to serve as the interface with themeasurement stage (or to transfer the bacteria into an IR-compatiblesolvent) (c) a measurement stage that measures the infrared signature ofthe bacteria in transmission mode, including other measurement modesknown to persons skilled in the art such as attenuated total reflection(ATR), diffuse reflection infrared Fourier transform (DRIFT), and othermodes which consists of the spectrometer (source and detector), flowcell or detection window, interface to provide digitized data foranalysis, and a waste collection stage, (d) a microprocessor or similarcomputer system to collect and digitize the acquired data, (e) ananalysis stage which comprises software to perform background and sampleassessment, background subtraction, deconvolution and other conventionaltechniques to determine attributes of the bacteria and a database (oraccess to a database) consisting of the spectra of bacteria referencematerial, (f) a reporting stage which enables transmission of resultsinto the desired format for comparison with databases, permits onscreendisplay, text messaging, and transmission and sharing onto otherperipheral devices such as a printer or hand-held device, and (g) acontrol stage that comprises software to controls the timing of thedifferent steps, power requirements, and parameters required at eachstep and phase of the present invention (such as fluid speed,centripetal force generated, scan speed, data acquisition rate, etc.).

In another embodiment, the microfluidic separation stage comprises adisposable mini-centrifuge or a spiral path and a fluid flow controlsystem (which is non-disposable). It is contemplated that the fluid flowcontrol system may be a vacuum pump or actuators for active and passiveflow control. It is also contemplated that the mini-centrifuge or aspiral path is capable of generating sufficient centripetal force toseparate the components of the sample matrix from the bacteria such thatthe bacteria are maintained intact. And the bacteria and the matrix(separated from the bacteria) are moved by the fluid flow control systemthrough all the other stages of a device according to the presentinvention.

In some embodiments, the mini-centrifuge or a spiral path furthercomprise a sub-system to lyse the components of the matrix. Otherembodiments comprise a component and means to mix the stream(s) from themicrofluidic separation stage with a carrier solvent.

Yet other embodiments comprise an interface with the measurementpreparation stage that helps transfer fluid from the microfluidicseparation stage to the measurement stage. Typically, the diameter ofthe channel in such an interface would be in the range of about 0.1× to10× relative to the diameter of the channel in the microfluidicseparation stage. The measurement preparation stage optionally comprisesa filter with a molecular size cut-off of about 0.45 microns or smallerand may be made of silicon, silicon nitride, polyethylene,polyvinyldifluoride, or Teflon® or other compositions known to personsskilled in the art.

It is contemplated that a carrier solvent or solution, when utilized,will be infrared (IR) compatible such as formaldehyde, n-hexane,ethanol, acetone. And the carrier solvent may contain some water in therange from about 0 to 90% by volume.

In other embodiments, the measurement stage comprises an infraredsource, beamsplitter, mirrors, beam condenser, objective, and detector.And it may further comprise a flow cell or a detection window to enablemeasurement. Preferably, the source is capable of emitting radiation inthe range from about 300 to 5000 cm−1, and may deliver coherent orincoherent light. It also may utilize a frequency comb in situationswhere a coherent beam containing a continuum of frequencies over one orseveral octaves are needed. The spectrum and power of these pulses areoptimized to improve signal-to-noise ratio and to enhance resolution.Applications utilizing such frequency combs include classical andultra-high resolution near-field microscopies. The infrared beam may befocused with the help of lenses and objective so that the beam widthoptimally ranges from about 1 micron to about 1 mm, as is commonly doneusing an FTIR microscope accessory. Preferably, the beam widthapproximates the dimensions of the microorganism being analyzed, forexample, bacteria.

In yet another embodiment, the detector is capable of detecting infraredabsorbance in the range from about 300 to 5000 cm−1. In a preferredembodiment, the detector comprises an array from about 1×1 to about256×256. Additionally, the detection window comprises a variable pathlength, and is made using IR-transparent material (such as silicon,calcium fluoride, potassium bromide, or zinc selenide).

Persons skilled in the art will understand that the measurement stagemay comprise various accessories that permit measurement intransmission, attenuated total reflection, and other reflection modes.Additionally, the detector preferably is located between 0.1 to 10000wavelengths. Detectors located within one wavelength can be used tomonitor the evanescent waves enabling inspection of the sample with veryhigh spatial and spectral resolution. This location of the detectorwould typically be utilized in near-field infrared microscopyapplications. Detectors located further away represent the moreclassical approach to infrared spectroscopy.

Other preferred embodiments comprise an analysis stage that utilizesfourier or fast Fourier transforms to convert the acquired data to aspectral signature. The analysis stage further utilizes the signal fromthe fluid flowing through the detection window over time to identify thebaseline signal or to determine changes to the initial signal. Thissignal may be, for example, at the frequency ranges where water,protein, peptidoglycan, or phospholipids absorb. Systems according tothe invention will distinguish between the baseline signal and thesample signal, and are capable of subtracting the baseline signals fromthe sample signals. Preferably the analysis stage utilizes secondderivatives and other techniques such as Gaussian deconvolution,auto-correlation, and Savitzky-Golay smoothing, which are well known toa person skilled in the art of analyzing and interpreting spectral data.

In another aspect of the invention, the analysis stage evaluates thespectral profile obtained from each sample signal to determine if itcould potentially be from a bacteria and labels such profiles or signalswithin a profile as bacterial signals. And the system determines whethera detected bacterial signal is attributable to, for example, a grampositive, gram negative, or gram variable bacteria. In preferredembodiments, the analysis stage determines the confidence (probability)of the determination. In certain embodiments, the analysis stagecompares the bacterial signals with spectra in a database to establishthe identity of the bacteria.

In other preferred embodiments, the reporting stage transmits the resultfrom the analysis stage to an output device of choice (for example, on ascreen, printer, USB stick) or transmits it, for example, via Wi-Fi,wired, Bluetooth or cellular modes to the destination of choice.

In yet another aspect of the invention, the measurement stage comprisesa waste collection receptacle that is sealed and will be disposedaccording to procedures determined by the user of the system.Optionally, the measurement stage comprises wash solvents and a washstep that washes the non-disposable surfaces that contacted the sample.The wash solvents may consist of a mixture of ethanol and water, waterand detergent, and pure water may be used to reduce or prevent samplecarryover from one measurement to the next. Optionally, the measurementstage may consist of the channel being overlaid on an accessory such asan ATR or a transmission cell or a channel consisting of pores (about 10nm to 1000 nm), which would enable filtrate smaller than these pores tobe separated from intact bacteria by filtration.

Briefly described, in a preferred embodiment, the present inventionprovides a means to detect and identify bacteria in any liquid samplematrix using a combination of microfluidic-based separation andspectrometry. The sample matrix in this embodiment is any matrix thatcontains bacteria.

The integrated device consists of a stage that separates the bacteria,for example, from the rest of the sample matrix and prepares it for themeasurement stage, which obtains the infrared spectrum of the bacteriaand the background. The spectra is analyzed to determine ifcharacteristic features associated with a bacteria are present andwhether the features provide additional features that permitclassification of the bacteria, for example, as gram positive, gramnegative, or gram variable, or on the basis of their genus, species,strain, or antibiotic resistance.

Preferably, Fourier transform infrared spectroscopy (FTIR) is utilizedto detect and identify the bacteria but other spectroscopic techniques(for example, Raman spectroscopy or mass spectrometry) can be utilizedas well. FTIR is a well-known analytical technique. The presentinvention enables microorganism identification and also diagnosis at thepoint of need and without a need for culturing a sample. It also enablesdisease diagnosis through identification of pathological bacteria at lowcost without utilizing antibody or nucleic acid sequence basedidentification.

Using this method, any sample matrix can be analyzed for the presenceand identity of the bacteria, if present, without the need forculturing. This dramatically reduces the time needed for analysis. Bynot using any antibody or nucleic acid primer for recognition, thedevice is low-cost, portable, and does not need additional laboratoryinfrastructure to support and enable its use at the point of need.

The present invention, in a preferred embodiment, is an integrateddevice that is capable of separating bacteria from a sample matrix,enriching the bacteria, obtaining the infrared spectrum of thebackground and the bacteria, subtracting the background spectrum fromthe bacteria spectrum, analyzing the resulting spectrum through knowndeconvolution techniques and reporting the results using peripheraldevices that are appropriate for the particular intended analyticalpurpose. The analysis of the spectrum may also involve comparing thespectrum of bacteria to that of a reference, which is stored in adatabase,

FIG. 1 provides a schematic that identifies the major components of thedevice according to the present invention, and identifies the majorfunctions of these components. In general, a sample 101 is obtained foranalysis. This sample is prepared by dilution or other steps asindicated by the sample dilution stage 103. Some of the components ofthe sample matrix 105 are lysed and intact bacteria are separated fromthe matrix components using a microfluidic separation stage.

Next, intact bacteria are provided to the infrared signal measurementstage 107. Data from this measurement are communicated to a spectralanalysis unit 109. And results of the analysis are reported viareporting stage 111. The entire process is designed to be a continuousprocess without any interruptions to the separation and measurement orrequiring any manual intervention.

All liquid flow is controlled and managed by micropumps or capillaryaction. The pump can use any of the commonly utilized driving forces forpumping fluid—piezoelectric, pneumatic, electroosmotic, etc. Followingthe treatment in the sample dilution stage 103, the entire sample istransferred to the sample matrix lysis stage 105 and the microfluidicseparation stage 105. The movement from one stage to the other iscontinuous and not performed in batch mode. The separation of the intactbacteria occurs as the fluid (containing intact bacteria and componentsof the sample matrix) moves through the microfluidic separation stageresulting in two outlet streams (shown in FIG. 1 ). The sample componentstream is collected in a waste receptacle and is normally discardedwhile the stream containing intact bacteria moves to the measurementstage 107.

The fluid containing largely intact bacteria moves through themeasurement stage where the IR signal is measured and produces aquantified digital signal. The fluid is collected in a waste receptacle,which is discarded. In certain applications, the analyte of interest(for example, cholesterol, glucose, etc.) may be present in the wastestream from the measurement or separation stage. In such situations, thewaste stream may also be routed to the measurement stage to measurethese analytes utilizing the same detection apparatus (FTIR).

The digitized data from the measurement stage is then transmitted to thespectral analysis stage 109. The transmission can be internal to thedevice, to a local computer or server and can be accomplished usingwires (for example, an RS232 connection) or wireless (Bluetooth orWi-Fi).

Following analysis of the digitized data (as described in the spectralanalysis stage 109), the results are transmitted to the same or otherappropriate peripheral device 111, again using a wired or a wirelessconnection, as needed.

Stages 103 & 105 are disposable and are discarded following analysis ofeach sample. The stages can handle either a single sample at a time ormultiple samples simultaneously. In the latter case, multiple channelsare utilized to move the fluid until the measurement stage. At thisstage however, the different channels are opened based on a timingsequence controlled by a conventional microprocessor so that the IRspectrum of one sample at a time is measured. All of the streams go tothe same waste receptacle in the end.

Sample Matrix 101:

The sample itself can be whole blood, or a food matrix as described. Inthe case of solid samples, a preparatory stage involving “liquefying”the solid sample, such as food, is needed. This can be accomplishedusing practices that are well known and practiced by food safetytesters. They typically involve blending or mixing in a Waring® blenderor a Stomacher® blender, such as the Stomacher® 400, and the Stomacher®3500.

Any container that is capable of holding liquids can be used as a samplecontainer/receptacle such as test tube, microcentrifuge tube, or asimple plastic container. The container is normally glass orpolypropylene though any biocompatible material that does not adsorbbacteria or the analyte of interest (such as other organisms,cholesterol, glucose, urea, etc.) can be used. The container sizedepends on the nature and volume of the sample and can range from 10 uLto 500 mL. The requirements of the container used are that: (i) itshould be capable of being sealed (to prevent contamination) and (ii) itshould have an outlet port that is compatible with the sample dilutionstage 103. In its preferred embodiment, the sample container is a venousblood collection tube such as, for example, a Vacutainer® tube. Thesetypically and preferably consist of a test tube with a tube top that canbe pierced and may contain agents such as EDTA to prevent bloodclotting.

The tube top is placed against the first stage (Sample Dilution Stage103) that contains a needle to pierce the tube top. The needle may bemade of stainless steel or other similar materials. In the case of othercontainers, an outlet from the sample container that forms a tight sealwith the sample dilution stage is needed. This connector can be made ofpolypropylene, polyvinyldifluoride, silicone, Teflon®, or anybiocompatible material that does not adsorb bacteria or the analyte ofinterest. The diameter of the connector can be varied depending on thevolume of the sample. In its preferred form, the diameter is greaterthan about 1 cm. Though smaller diameters can be used, the diametercannot be smaller than 200 microns. The length can be varied dependingon the conditions of the measurement. A key requirement is that thehold-up volume in the connector tube should be approximately one-tenththe sample volume or less.

A fluid control device (such as a micropump) preferably will be used tomove the sample through the different stages. This can be positionedeither beside the sample receptacle (preferred) so that it pushes theliquid through the intended flow path or beside the waste receptacle sothat it pulls the liquid through the intended flow path.

Gravity may be used to assist the fluid flow and prevent air bubblesfrom entering the fluid flow path. To achieve this the sample receptaclemay be placed at a higher elevation relative to the next stage.

Sample Dilution Stage 103:

The sample enters this stage via the connector/needle. From this pointuntil the waste receptacle, the system is sealed to prevent/reducechances of external contamination. A one-way vent optionally may beprovided to ensure that a vacuum does not form within the system. Inaddition, a degasser component optionally may be added to ensure asmooth flow.

Sample components refer to different elements of the matrix depending onthe sample. For example, in the case of identifying the bacteria presentin blood, the sample components include red blood cells, white bloodcells, plasma proteins, platelets, and other blood factors. In the caseof food, the components include elements that constitute particles offood or its constituents (such as fatty acids, fats, lipids, proteins,etc.).

In this stage the sample is diluted to ensure that the viscosity of thefluid is similar to that of water and filtered to exclude very largecomponents in the sample matrix.

A diluent preferably water (but which can also be phosphate bufferedsaline or other biologically acceptable buffer) is utilized fordilution. The volume of diluent added is to ensure that the viscosity isreduced to be similar to that of water (1 centipoise). For example, thetypical viscosity of blood is approximately 10 centipoise at 20° C. Thehigh viscosity of blood is largely due to the presence of hematocrit andthe relationship between hematocrit and viscosity is well known. [3]Diluting blood in the range of about 5 to 50-fold (preferred target isabout 10-fold) will reduce the viscosity to the desired level ensuringthat the flow of the sample through the system is not affected due toincreased viscosity.

The diluent is typically stored in a bottle or bag made of glass orplastic (polyethylene) with a seal that can be pierced or with an outlettube. The bottle, in its preferred form, is stored inverted and thediluent is added to the stage via the needle/connector. The material ofconstruction and considerations are similar to those described for thesample receptacle. The addition of the diluent may occur due to gravity(preferred) or via a fluid control device. Optionally, a deviceaccording to the present invention may include a hook or other elementto hold a fluid reservoir. Optionally, the sample is filtered followingdilution.

A sequence of filters with varying pore sizes may be used to preventclogging downstream. These filters will all possess a pore size greaterthan the size of the bacteria (generally 2-10 microns) when bacteria arethe intended organism or analyte to be identified, but are preferablygreater than about 100 microns, which should permit passage of theanalytes and restrict matrix material. The filters can be constructed ofcommonly used materials such as polyethylene, polyvinyldifluoride, orTeflon®, though other materials may also be considered as will be knownto persons skilled in the art. The diameter of the filter can vary from1 cm to 15 cm. Large volumes of sample (for example, greater than about25 mL) will require a larger diameter filter. The size of the filter canbe determined by one of the three traditional methods used to sizefilters—Volume endpoint (Vmax), Pressure endpoint (Pmax), and turbidityendpoint (Tmax). The preferred method is the Vmax method. The sampleflow direction can be tangential to the filter surface or longitudinalto the filter surface. The former is the preferred embodiment to reducethe chances of filter clogging due to the components of the samplematrix.

Fluid flow to the next stage occurs via a manifold (preferredembodiment). While a connector of short length (less than about 10 cm)could be utilized, a tightly sealed manifold connecting the two stagesis preferred. The diameter of the manifold optimally will range fromabout 100 microns to about 10 cm. The manifold, in its preferred form,is made from a thermosetting plastic that is analyte-compatible (that isdoes not adsorb or interact significantly with the analyte, such asbacteria).

Microfluidic Separation Stage 105:

The separation stage is a disposable component that is intended to besingle-use. The separation stage can accommodate analysis of a singlesample or multiple samples. A number of methods and approaches are knownand utilized for making microfluidic devices such as [4-6]. Theseparation stage consists of two sections—a sample lysis 105A sectionand a separation section 105B. While the presence of the lysis sectionis optional, the preferred embodiment includes both sections. Thepurpose of the separation stage is to isolate and concentrate intactbacteria for infrared analysis.

FIG. 3 shows the schematic of the preferred embodiment of the separationstage 105. In the preferred embodiment, large non-bacterial cells (suchas red blood cells) are first separated from intact bacteria usinginertial focusing. This is followed by selective lysis of the remainingnon-bacterial cells (in the preferred embodiment, by the use of porousmonoliths) resulting in fluid containing intact bacteria andnon-bacterial cell debris. The debris is then separated from intactbacteria using differences in hydrodynamic size. In the preferredembodiment, this is achieved by deterministic lateral displacement. Thisstage also reduces the total volume of the fluid that moves to thefiltration stage. In the preferred embodiment, the intact bacteria isreadied for infrared analysis by filtering the fluid using ultrathinsilicon membrane filters, which eliminates water and any remaining lysisdebris leaving intact bacteria on the surface of the filter, which canbe analyzed by any appropriate technique. In the preferred embodiment,this technique is infrared spectroscopy. The entire separation andconcentration is accomplished without the use of reagents such as DNAsequences or antibodies.

The separation stage can be connected to the stages preceding (103) andfollowing (107) using connectors or manifolds (preferred). These can befabricated using any thermoplastic material that is compatible with theanalyte or glass. The preferred materials of construction arepolycarbonate and cyclic olefins. Examples of the choice of materialsand methods for forming the manifolds and connectors have been describedin the literature, for example [7]. The inner diameter of these tubingcan be in the range of about 0.001 mm to about 10 mm with a lengthranging from about 0.1 mm (manifold) to about 1 m. A shorter length isalways preferred.

Lysis Section 105A:

Following filtration and before reaching the separation stage, anotherprocedure, as discussed below, is applied to disrupt non-bacterialcells. This is likely to be needed when dealing with whole blood, whichcontains red blood cells, white blood cells, and platelets.

The disruption of the non-bacterial cells can occur through mechanicalmeans (preferred) relying on shear stress to lyse the cells or bychemical means. For disruption by shear stress, a sample is typicallyforced through microporous surfaces that contain pores with a diameterof about 1-10 microns (for instance resins used in chromatography). Themovement of cells such as white blood cells, red blood cells, andplatelets through these pores creates a high shear force, which in turn,causes these cells to be lysed. However, bacterial lysis to asignificant degree will not occur due to the more robust nature of thebacterial cell. Alternatively, chemicals such as polysorbates, tritonX-100, sodium cholate, or tri(n-butyl)phosphate, may be used to disruptcell walls by chemical means.

In the preferred embodiment, the lysis section 105A consists ofmicrochannels packed with resins of the desired pore size and the sampleis forced through these pores at a flow rate ranging from about 20microliters per minute to up to 1 milliliter per minute (preferred isabout 0.5 milliliter per minute). The channel width, height, and lengthare greater than about 100 microns, about 50 microns, and about 5 mmrespectively. While a straight channel with the above dimensions ispreferred, curved channels or channels that double back among themselves(to increase length of the channel without increasing the overall lengthof the stage) can also be utilized.

The materials of construction and fabrication of the channels aresimilar to those utilized for the separation section 105B describedbelow.

In cases with a large load of non-bacterial components that are ofsimilar sizes bacteria (for example, blood), an additional step prior tothe lysis step may be added. The function of this step is to reduce thenon-bacterial cells load prior to lysis in order to improve lysisefficiency and reduce the potential for clogging. Reduction of bloodcells in the samples will be accomplished using inertial focusing takingadvantage of the size difference between bacteria (1-5 μm) and red/whiteblood cells (7-15 μm). Of the various inertial focusing approaches, theuse of spiral microchannels has been demonstrated as a uniquelyefficient method to separate cells that are similar in size with highthroughput and separation efficiency. [8] Spiral channels fabricatedusing thermoplastics will selectively reduce the concentration ofnon-bacterial cells such as red/white blood cells. The design andfabrication of the spiral channels are similar to those described inSeparation Section 105B (Spiral path embodiment).

Fluid exiting the lysis stage is now a largely aqueous system, whichconsists of intact bacteria and multiple components, such as proteins,nucleic acids, fragments of cell walls and cell organelles. Thesefragments are typically smaller in size than the intact bacteria. Thisstage minimizes or eliminates non-bacterial components that are largerthan the bacteria with an efficiency greater than about 80%. The samecontinuous channel without any resin serves as the inlet channel for theseparation section 105B.

Separation Section 105B:

A preferred configuration for the separation section 105B is to flow asample via a microchannel that traces a spiral path (“Spiral path”embodiment described below). Separation may also be achieved usingdeterministic lateral displacement (DLD), [9], [10] in which a lineararray of microposts will serve to focus and direct larger rigid bacteriaparticles to an outlet channel while shunting smaller and flexible celldebris to a waste outlet. This separation method will also provide theadded advantage of significantly reducing the total collected fluidvolume. While the this section can be constructed using many differentmaterials, the preferred material is a thermoset plastic, which can beeasily fabricated at low cost and is generally compatible with food andbiological matrices. The channels (spiral path, described below) can bemanufactured using molding, photolithography, or micro-machining methodsas are well known to those skilled in the art of microfluidic devicefabrication. Any of these approaches can be adapted for producing theseparation section described in this invention.

While microfluidic devices have reduced the volume of reagent needed andincreased the speed of the analysis, they are usually able to handleonly small volumes of sample. Thus, if the concentration of the analyte(for example, bacteria) is low, the signal from the analyte may be toolow to be detected reliably, let alone used for identification. One wayto improve the concentration has been to use a technique like PCR toamplify the analyte (nucleic acid) or to immobilize antibodies in orderto concentrate the analyte.

In the present invention, by way of contrast, the separation andconcentration is accomplished using physical forces, for example,inertial or centrifugal forces. Thus, there are no zones where theanalyte has to contact a reagent in order to enable concentration ordetection and relatively faster flow rates can therefore be utilized.This approach also enables processing of a large volume of sample which,in turn, decreases the lower limit of detection compared to othermicrofluidic devices.

The objective of the separation section to separate intact bacteria fromthe rest of the sample matrix and enrich it. The section relies largelyon inertial/centrifugal forces to accomplish this task.

The inertial/centrifugal force can be generated by forcing the liquidthrough channels that describe a spiral fluid path, DLD, as discussedbelow in a preferred embodiment, or by a mini-centrifuge. The spiralmicrochannels & DLD microposts are fabricated on a microfluidic chip.

The magnitude of centripetal force or acceleration is related to themass of the different components of the sample matrix. Thus, componentsthat are smaller in mass compared to the bacteria will experience asmaller force relative to the bacteria.

The fluid flow is introduced to the spiral using the outer arm of thespiral (preferred embodiment). Alternatively, the fluid can also beintroduced at the center of the spiral. If a mini-centrifuge is used,the fluid is normally introduced at the bottom of the centrifuge.

The Spiral Path Embodiment:

The path preferably is fabricated on a glass, PDMS, cyclic olefins,polycarbonate, or similar surface that has been used for preparingmicrofluidic chips. The process for fabricating such a path may be alithographic technique. However, other processes are well known,understood, and utilized in the fabrication of microfluidic devices, asdescribed before, and can also be employed.

The spiral may be a two-dimensional or three-dimensional spiral and canbe any of the different types of spirals that have been mathematicallydefined (such as Archimedes spiral, Fermat Spiral, logarithmic spiral,etc.). A preferred spiral path is the Archimedes spiral.

The parameters of the spiral (usually radius and angle) can be furtheroptimized to improve the efficiency of the separation as can routinelybe accomplished by persons skilled in the art.

The fluid entering the spiral path is preferably discharged into theoutlet (where the bacteria are being directed) or a discharge channel(to collect the sample matrix components).

The outer edge of the channel optimally will have openings that lead toa separate discharge channel. The openings can be rectangular, circularor any shape with an effective diameter range from about 10 microns toabout 250 microns. The channel too can be of any shape (rectangular ispreferred) with a width greater than about 50 microns and a heightbetween about 10 to about 500 microns.

The sample from the lysis section 105A enters the separation section atthe outer arm of the spiral with a fixed velocity. The spiral shape ofthe flow channel causes the components that possess a lower mass thanthe bacteria to be directed to the discharge channel while the bacteriastay in the spiral fluid path until it they reach the exit of thespiral.

At the time of exit, the bacteria have largely been separated from most,if not all, of the other sample components. A reduction in the level ofthe sample components of greater than about 80% is achieved. Thebacteria are in a largely aqueous medium.

This section has an interface that enables it to be locked in placeagainst the measurement stage enabling a continuous fluid flow pathbetween the separation and measurement stages as well synchronization ofthe steps (sequence and timing) taking place in the two stages.

Deterministic Lateral Displacement (DLD):

DLD uses flow through an organized array of “obstacles” (microposts) toseparate particles based on their hydrodynamic size. It does this byselectively influencing the trajectory of particles above a certainsize. The fundamental principles explaining this phenomenon have beenknown for close to a decade and have been modeled by several researchgroups [11-13]. These devices can be fabricated using standardlithographic techniques. Typical materials used to fabricate DLD devicesinclude silicon and PDMS. The array of microposts can also be directlyembossed onto thermoplastic devices.

The key variables that influence the separation include the size of themicroposts, gap between microposts, and offset between two rows of thearray (sometimes referred to as tilt angle relative to the fluiddirection). In the preferred embodiment, the diameter of the cylindricalmicroposts may range from about 10-500 microns, the gap between themicroposts can range from about 1-200 microns, and the offset can rangefrom about 0.1 to 5 microns. The total length of the array ranges from 3mm to 20 cm. Multiple arrays may be arranged in sequence to provide highresolution separation of the bacteria from the lysis debris. Othergeometries for the micropost, such as I-shape, can also be used toaccomplish the separation as described by Zeming et al. [14]

The inlet to the DLD is the same as the outlet from the previous stage.An additional input that permits addition of carrier solvent (such aswater) may also be utilized. The design of the DLD ensures that theintact bacteria are pushed away from the main fluid stream (whichcarries the lysis debris). This “side stream” carrying the intactbacteria is the outlet that is directed to the next stage (infraredsignal measurement stage 107). The main stream containing the lysisdebris in the preferred embodiment is discarded. But this stream mayalso be utilized for further analysis. Reduction in lysis debris levelsgreater than about 95% is expected. The volume of thebacteria-containing stream is expected to be approximately one-hundredthof the initial volume entering this stage.

The use of DLD to separate RBCs from whole blood has been reported, forexample by Zeming.[14] However, their application of DLD is effectivebecause the size difference is large between RBCs and other blood cells.When it comes to separating bacteria from RBCs, white blood cells(WBCs), and platelets, the size differences are not as large.Inefficient separation of bacteria from other cells will increase theuncertainty in identification accuracy. This is because these othercells would contribute infrared signals of their own, if present, thusinterfering with accurate identification.

The present invention optionally includes a selective lysis step, whichhas been found to provide a major advantage in the rapid detection ofmicroorganisms, because it now “creates” a large size difference bylysing all cells other than bacterial cells. Following lysis of theblood cells, when applicable to a particular sample, the bacteriabecomes the largest entity and so can be separated appropriately fromthe rest of the fluid.

Mini-Centrifuge:

The centrifuge preferably is constructed using a polymer such aspolypropylene, polycarbonate, or Teflon®. The design and principlesunderlying centrifugation and its use for separating cells and organismsfrom various matrices is well known. Examples of such informationinclude Industrial centrifugation by Leung [15] Centrifugation inBiology and Medicine [16], and Continuous centrifugation in virusprocessing [17]. The maximum capacity of the designed centrifuge isintended to be less than about 500 mL. It is a single-use component andis discarded following sample preparation.

The mini-centrifuge has an inlet channel that for introducing the sampleinto the centrifuge (that is, the channel from the lysis section 105A),an outlet channel for transporting the intact bacteria to themeasurement stage 107, and a discharge channel for the rest of thecomponents.

The key variables influencing the efficiency of separation have beendescribed in various well-known texts describing centrifugationprocesses. The speed of rotation, height of the mini-centrifuge, angleof the walls of the centrifuge from the vertical, and duration of thecentrifugation are some of the key variables. These variables can beadjusted routinely by persons skilled in the art to optimize theseparation of analyte (such as bacteria) from matrix (such as blood orenvironmental sample sources).

The centrifuge preferably is rotated by a spindle, which is attached toa motor. The motor in turn is powered by electricity (AC/DC) or by abattery. The preferred power source is a battery.

Infrared Signal Measurement Stage 107:

The spectral region of interest where bacteria have been known togenerate characteristic infrared signatures is in the mid-infraredregion (about 4000 cm−1 to about 400 cm−1). Spectroscopic methods ofanalysis generally are known to be rapid and can be automated to testmultiple samples at a time. This aspect of spectrometry preferably isutilized in combination with a microfluidic separation stage to enablethe detection of bacteria from any sample matrix. The bacteria could beany species of interest. Some samples of bacterial species includeSalmonella species, Listeria species, and Streptococcus species.

The primary basis for the detection of the bacteria by the preferredmethod of detection, FTIR, is the characteristic infrared spectrum ofbacteria in general and the difference between species and strains,specifically. The specificity in identification by the devices andassociated methods of the present invention is due to the nature of theconstituents of the bacterial cell wall—proteins, polysaccharides,nucleic acids, and phospholipids. It is also postulated that othercomponents of the bacteria (such as DNA) and organelles ofmicroorganisms may also contribute to the infrared signature. Thecomposition of the cell wall, for example, affects the intensity andfrequencies of the IR absorbance pattern and thus yields characteristicinfrared signatures that enable both detection and identification of thedifferent bacteria. In principle, any intact organisms can bedistinguished from each other on the basis of their IR signature,provided a satisfactory separation from other components has taken placeprior to the measurement and the organism has been sufficientlyconcentrated as to be above the limit of detection.

The analyte enters the measurement stage 107 via a manifold similar tothat described before. At this stage, the analyte largely containsdiluent and bacteria. The concentration of the bacteria in the fluidentering the measurement stage 107 preferably is greater than or equalto 10-fold higher than that in the original sample, the volume of thefluid of the original sample is also reduced by an equal or largeramount, and most of the components of the original sample matrix,especially the relatively larger-sized components, are lower (less thanabout 10-fold) than in the original sample. For example, an initialsample volume of 1 mL may go through the various steps described in theprevious sections resulting in bacteria-containing fluid that isconcentrated and/or filtered within an area ranging from about 0.01-0.3mm². The ability to process a large volume of sample and reduce it to asmall area of measurement while recovering most of the bacteria enablesdetection and identification of the bacteria without culturing. Theseparation stage (105) plays an important role in isolating the intactbacteria and removing most, if not all, of the substances that mightinterfere with the infrared signal acquisition. Additional concentrationof the bacteria (by the methods described below) in the measurementstage (107) aids in improving the sensitivity of the measurement.

In the preferred embodiment, the intact bacteria following separation asdescribed in 105B flow along a channel (made of the same materials ofconstruction and dimensions as described in section 105A and 105B) untilit reaches a zone (referred to as IR measurement zone) where theinfrared measurement is made. This zone is constructed using an IRtransparent material such as silicon and is positioned so as to be inthe infrared beam path.

This zone preferably is a filtration channel fabricated using ultrathinpolycrystalline silicon such as the one described by Striemer et al.[18] The aspect ratio of the filter (diameter of pores to thickness ofthe filter) is approximately 1.0 (the aspect ratio preferably can rangefrom about 0.001 to 100). Flow through this channel enables anyremaining small-sized debris and diluent to be filtered leaving theintact bacteria on the surface of the filter. The filtration channelwidth can range from about 0.1 to 1 mm, while the length can vary fromabout 0.5 to 5 mm. The pore diameter can range from about 20 nm to 2microns, though diameters less than about 1 micron are preferred inorder to retain a large percentage of bacteria. The use of the ultrathinfilter is a critical step since it permits measurement in transmissionmode at a good s/n ratio, which improves sensitivity. Conventionalfilters are too thick and generally have a broad pore distribution. Theultrathin filters have a narrow pore distribution. Together the twofeatures also enable a higher filtration rate, a lower pressure drop,and lower loss of intact bacteria.

Other options, such as an infrared flow cell or an ATR (Attenuated totalreflectance) accessory, can also be utilized, as described below.

The measurement stage detects and measures the infrared signature of thebacteria in transmission, attenuated total reflection, reflection, andother modes and consists of the spectrometer (source and detector), flowcell or detection window, interface to provide digitized data foranalysis, and a waste collection stage.

The measurement itself is made in a manner similar to that used forinfrared measurements for different applications such as analytical andforensics (for example, trace residues, environmental contaminants,etc.). The instrument relies on interferometry to acquire aninterferogram, which is then subjected to Fourier transformation toyield the spectrum in the frequency domain. This type of dataacquisition has been well described in many textbooks on the subject ofinfrared spectroscopy such as Griffiths [19].

The source and detector and interface to provide digitized data foranalysis is similar to that utilized in other infrared spectrometers.The detector may also be a detector array (described below). The sourcemay be a regular thermal source or a laser-based source and may utilizecoherent or incoherent radiation that is capable of emitting radiationover the range from about 400 to 4000 cm−1.

The fluid from the separation stage may be diluted using a non-aqueousinfrared transparent solvent. This could be performed to reduce theeffect of the infrared signal due to water depending on the desiredsignal-to-noise (sn) ratio. When the ultrathin filter is used, this stepcan be avoided since most, if not all, of the signal due to water iseliminated due to removal of water by the filter.

The bacteria-containing fluid (bacfluid) can be readied for measurementin multiple ways—filtration followed by measurement of the bacteriaretained on the surface of the filter (preferred option), flow past theIR sensing zone (IR sensing zone option), flow through a flow cell (flowcell option), or use of an ATR (ATR option). In all cases, there iseither spatial or temporal (or both) resolution of the sample enablingdiscrimination between signals from different bacteria as well asbetween bacteria and the background. Previous attempts, such as Helm etal. [1], [20], to measure IR spectra of bacteria did not possess any ofthe components described in this invention and consequently measured theweighted average of all signals. This resulted in the need for largeamounts of highly purified bacteria (greater than about 10⁴ CFU/mL). Theapproach described in this invention does not require such a largequantity of bacteria as in prior approaches and devices, and alsoprovides a higher sensitivity. Various different options for alternativeembodiments are described below.

In the case of filtration, the bacteria-containing fluid (referred to asbacfluid) is passed through an IR-compatible filter (such as ultrathinsilicon/silicon nitride filters from SimPore or polyethylene filtersfrom Millipore) that has a pore size less than about 0.5 microns(preferred size is about 0.2 microns or less). The bacteria are retainedby the filter while those components that were smaller than the bacteriaand not eliminated by the separation stage will pass through the filter.This filter is referred to as the bacteria filter.

The filter may be of any shape (rectangular, circular, etc.) covering anarea ranging from about 100 micron squared to about 10 centimeterssquared.

The bacteria on the filter are illuminated by the IR light source (forexample, by globar, tungsten filaments, or lasers from Block Engineeringor Lasnix) while the readout is performed using a single point detectoror an array detector. The single-point detector provides the weightedaverage of the infrared signals in the measurement area (e.g. about100×100 micron). The detector array is arranged such that it covers theentire surface of the filter. Infrared radiation from specific locationsof the filter surface is gathered by the array providing spatialresolution and a spectroscopic image of the filter surface. This, inturn, enables better discrimination of the bacteria from the filtersurface and other moieties that may be on the filter surface. The arraycan be about 1×1 to about 1024×1024. The detection can measure eitherthe near-field far-field infrared signals.

A reference filter may be utilized in order to obtain a backgroundspectrum. This reference filter is placed after the first filter and isidentical with respect to size, dimension, and material of construction.The fluid that passed through the bacteria filter passes through thereference filter as well.

The IR sensing zone preferably is made of silicon and integrated withthe exit channel from the separation section 105B. The measurementpreferably is made in transmission mode. The silicon may be coated withanti-reflective coating to improve the transmission percentage.Alternatively, silicon crystals to enable ATR measurements may also beutilized as will be known to persons skilled in the art. The methods ofintegrating the IR sensing zone to perform FTIR measurements is wellknown and has been described earlier for monitoring reactions inmicroreactors. [21-23]

The exiting fluid can flow through the IR sensing zone (Flow throughmeasurements) or may be analyzed in batch mode. In batch modemeasurements, the fluid is typically introduced as tiny droplets (volumeranging from about 1 nanoliter to about 100 microliters). These dropletsare dried either by flowing a drying gas such as nitrogen or by allowingfor environmental conditions to permit drying. The result is that thebacteria form a thin film (ideally a monolayer) on the surface of the IRsensing zone with little to no contribution from water in the IRspectrum. Such a thin film can be analyzed in transmission mode or usingalternative measurement modes such as ATR, DRIFT, etc. Such methods ofspotting or microprinting of bacteria is well known [24]. However,earlier approaches utilized pure culture for such spotting, whereas thisinvention seeks to eliminate the need for pure culture.

The sensing zone area ranges from about 2-50 millimeters in length,about 10 to 500 microns in width, and about 6 to 1000 microns in height.

Flow-Through Measurements:

In this case the bacfluid flows through a channel at a fixed velocitywhile transiting the detection window. The channel can be etched onto asample accessory (such as ATR) or may be constructed using knownIR-transparent materials (such as zinc selenide or silicon). The meansto construct an ATR accessory and obtain infrared measurements usingthis accessory is well known ([25] and [26]). The difference in thisinvention is that microchannels are imprinted on the surface of the ATRusing IR-transparent materials so that the fluid moves along a fixedpath. The ATR itself is constructed using established methods andtechniques. The microchannel path on the surface of the ATR can be astraight channel, a curved path, a sawtooth pattern, or any pattern.Methods of preparing such imprinted microchannels have been describedpreviously [27] and [28]. The width of the channel must be greater than50 microns to enable the bacteria to move through the channels. Dataacquisition is not affected by this change.

In another embodiment, the channel is manufactured by microboring a pathin an IR transparent material such as calcium fluoride and from a singlepiece. The latter requirement is to ensure that the interior surface iscontinuous without any cracks or deformities. The fluid flow path is astraight path through the IR cell though other forms such as curved orsaw tooth patterns are possible. An example of the construction of theflow cell and its use in acquiring data is provided by U.S. Pat. No.5,521,384 [29] and U.S. Pat. No. 4,618,769 [30].

The IR measurement can occur either at a fixed location in the flowchannel or may cover a region of the flow channel. In either case, thebacfluid is illuminated by the IR light source while the readout isperformed using a detector array. The array can be about 1×1 to about1024×1024. The detection can measure either the near-field or thefar-field infrared signals. This approach also enables background signalmeasurement, resolution between background and sample signals as well assignals from different bacteria.

Spectral Analysis Stage 109:

The spectral analysis is performed using a processor (or multipleprocessors) along with associated software. The software, in thepreferred embodiment, subtracts the background spectrum, compensates forthe presence of atmospheric carbon dioxide and water vapor. Theintensities at each wavelength are used to create a matrix of valuesfrom which various attributes are identified and utilized in theanalysis described below. The attributes include peak maxima positions,peak widths at specific wavelengths, peak minima positions, andpositions of minimum peak intensity. These attributes are also used inthe comparison against spectra in the reference database to determinethe closest match to the acquired spectra. Such spectral analysis andcomparisons are being commonly utilized in various applications such asbiometric identification, peptide map comparisons, vegetation mapping,etc. In the preferred embodiment, this analysis is performedautomatically without any manual intervention. However, the option toconduct a manual verification of the analysis and conduct otherassessments is provided.

Appropriate data technology is discussed below and in various referencematerials that are identified in this specification. It is contemplatedthat the processor may be part of a discrete computer device or systemor may be part of the device hardware, or is a combination of both, oris otherwise accessible for purposes of the present invention. Thesoftware consists of a set of computer-executable instructions and mayalso include data structures capable of storing spectral information.The software may be part of a computer, part of the device hardware, ora combination of both. These components are quite well known andcommonly utilized in acquiring data from various instruments. Examplesdescribing such systems can be found in [31], [32], and [33].

The data structure for a spectrum typically consists of theinterferogram and its Fourier transformed version. The Fouriertransformed data set normally consists of intensity measured at eachwavelength and enables one to trace the spectrum similar to the spectrumshown in FIG. 2 . Additional attributes of the spectrum such as baselinenoise, width of peaks, second derivative, and other mathematicaltransformations may also be stored as part of the data structure for asample. Normally the data structure is an “m×n” matrix where the “m”rows represent the number of wavelengths where measurements were madeand the “n” columns represent the different attributes of interest.

The data structures can also be part of a database management systemwith the option to search, query, and retrieve spectral information. Thedatabase management systems can be any of the well-known systems such asMySQL, Oracle, Microsoft Access, etc. The information stored in the datastructures normally contains spectral information of different bacterialspecies in different matrices.

Preferably, two (or more) databases are utilized. One of the databasesis a reference database, which contains spectral information ofbacterial reference standards acquired under controlled conditions. Theinformation in this database is used as a training set for validatingthe model as well as for comparison purposes. Databases available atsources such as the Institute for Microbiology at Munich TechnicalUniversity are utilized and may be augmented with new informationgathered by the user for the above purpose. The information in thesedatabases identifies factors that contribute towards predictive modelingfor determining the identity of the bacteria. Predictive models arebased on data cluster investigation techniques such as principalcomponent analysis (PCA), and hierarchical cluster analysis (HCA) and onclassification algorithms such as K-nearest neighbors (KNN) and softindependent modeling of class analogy (SIMCA). Such techniques areroutinely utilized by commercially available software such as PIROUETTEand JMP. Following training using known bacteria, the predictive modelis validated using additional known bacteria before utilizing the modelfor analyzing samples where the presence of bacteria and their identity,if present, are not known. In the preferred embodiment, the referencedatabase is provided along with the instrument. In addition, thevalidation using the reference database and a predictive model are alsoprovided. The user has the option to update or modify the referencedatabase and validation model. It is expected that this might generallybe done as the user obtains the spectra of additional bacterial speciesand strains or if the user has bacterial standards that may not be partof the original reference database.

The second database is typically a sample database that can vary fromuser to user and contains spectral information of all samples analyzedalong with information such as date and time of acquisition, location,sample ID, analyst, and any sample other tracking information desired bythe user. This database is utilized for trend analysis, review ofresults and preferably be maintained, backed up, and updated by theuser. The user may choose to maintain different databases for thedifferent departments, divisions, or laboratories. Thus, multiple sampledatabases may exist at a user site or on the device itself.

If the carrier solvent that is not water is utilized, one means torapidly screen for the presence of bacteria is to screen for thepresence of a characteristic “water” peak spikes in the sample. Thewater peak arises owing the presence of water within the bacteria andbecause this concentration of water is different from that of thesurrounding solvent.

The first step in the analysis is the subtraction of the backgroundspectrum. In the case of the detector array, this will be a regionwithout any absorbance in the regions where bacteria might absorb or aspectrum obtained from the reference filter. In the case of the flowthrough measurement, this will be a region without any substantialabsorbance in the regions where bacteria might absorb.

Following identification and subtraction of the background spectrum fromthe sample spectrum, the analysis (performed by the software) determinesif the spectrum possesses features that are common to all bacteria.These include possessing absorbance in the five major absorbance regionsin an IR spectrum. These are about 3000-2800 cm−1 (region 1), about1700-1500 cm−1 (region 2), about 1500-1200 cm−1 (region 3), about1200-900 cm−1 (region 4), and about 900-700 cm−1 (region 5). Region 5 isthe fingerprint region that contains weak but very unique absorbancesthat are characteristic to specific bacteria.

If the levels of bacteria in the sample are low, the absorbanceintensities will be low (despite the separation and enrichment).Therefore, each result determination made be the software may beaccompanied with a probability estimate that the result is accurate. Theprobability estimates are based on the predictive modeling software (theclassification algorithms), which assess the similarity of the spectralparameters of the unknown sample to that of the known samples. Some ofthe approaches utilized for predictive modeling were described earlier.Typically the algorithms used for such modeling compute an index thatmeasure the similarity of the data for the unknown sample to abenchmark. Examples of such indices include the Rand measure, F-measure,and Jaccard index. The indices normally serve as a measure of thepercentage of correct decisions made by the algorithm. In the preferredembodiment, the index reports the measure of the correct decisions madebalanced by the contribution of false negatives during the validation ofthe model. The estimate may be a number or represented by a color code,or graph. Typically, the expected probability for reporting a positiveresult is greater than about 60%, while greater than about 90% ispreferred.

Once bacterial presence in the sample is ascertained, the softwareproceeds to the next step in the analysis. If no bacteria are determinedto be present, the analysis terminates and the result is provided to theuser.

The next step in the analysis is the determination if the bacteria aregram positive or gram negative. Gram-positive bacteria possess a higherlevel of peptidoglycan (greater than about 50%) compared togram-negative bacteria. In addition, they also possess teichoic acidsand lower lipid, protein, and lipopolysaccharide content. Thesedifferences result in an infrared signature that is different from thegram-negative bacteria. If needed, the spectrum may also be comparedagainst a reference spectrum of gram positive and gram-negativebacteria. The reference spectrum is normally stored as a data structure,which could be part of the device hardware, a local computer, or storedon a server. During the comparison, the software queries the databaseand searches the data structures containing the spectra of bacteria (orother analyte) looking for the closest match to the spectrum of thesample. The comparisons are based on the intensity, frequency, and widthof the infrared absorption bands typically. If a match is found, thereference spectrum that matches the sample spectrum is temporarilyflagged in case a review by the user is needed. The recently acquiredsample spectrum is added to the sample database and utilized in variousanalyses such as trend analysis at the user's discretion.

As before, the determination regarding gram positive or gram negativemay be accompanied with a probability estimate concerning the accuracyof the result. In case a suitable distinction between gram positive andgram negative cannot be made, the result being reported as beingpotentially gram variable or indeterminate. The expected probability forclassification as gram positive or negative is greater than about 60%while greater than about 90% is preferred.

Following this stage of analysis, the next stage is the identificationof the species. In this case, spectral information is evaluated usingthe predictive modeling described earlier. In addition, the spectrumwill be compared against the individual species that are in thedatabase. An efficient search algorithm that enables comparison of thesample spectrum against the reference spectra is utilized to determinethe identity. If a suitably high degree of match (greater than about70%, while greater than about 90% is preferred) with a referencespectrum is achieved, the probable identity of the bacteria is reportedalong with the estimated probability of result accuracy.

Sample can be compared directly against the spectrum of a referencebacteria sample through visual examination or compared after standarddeconvolution techniques that are well known and frequently utilized inFTIR data analysis (such as Gaussian deconvolution or second derivativeanalysis) and which have been described by reports and books such as[19], [34], [35]. Second derivative analysis for instance, is used todetermine the position (for example, wavenumber) of peaks (usuallypoorly resolved peaks). The positions of these peaks are comparedagainst the reference to determine the closeness of match. Thecomparison of the sample spectrum against the reference spectrum isperformed as described earlier. To ensure reproducibility inidentification, combinations of the above approaches may be utilized.

Result Reporting Stage 111:

The results (presence of bacteria, gram staining classification, andidentity) are transmitted to the desired output format—on screen, USB,text message, and/or other peripheral devices such as a printer. Theparameters involved in the analysis along with the original data andestimations are stored on the local hard drive or transmitted to asecure location of the user's choosing. In industries where needed, afeature that tracks edits, analyst, and other pertinent information(referred to as “audit track”) is also provided.

Computer Systems:

Implementations of the various techniques described herein may beimplemented in digital electronic circuitry, or in computer hardware,firmware, software, or in combinations of them. Implementations mayimplemented as a computer program product, for example, a computerprogram tangibly embodied in an information carrier, for example, in amachine-readable storage device or in a propagated signal, for executionby, or to control the operation of, data processing apparatus, forexample, a programmable processor, a computer, or multiple computers. Acomputer program, such as the computer program(s) described above, canbe written in any form of programming language, including compiled orinterpreted languages, and can be deployed in any form, including as astand-alone program or as a module, component, subroutine, or other unitsuitable for use in a computing environment. A computer program can bedeployed to be executed on one computer or on multiple computers at onesite or distributed across multiple sites and interconnected by acommunication network.

Method steps may be performed by one or more programmable processorsexecuting a computer program to perform functions by operating on inputdata and generating output. Method steps also may be performed by, andan apparatus may be implemented as, special purpose logic circuitry, forexample, an FPGA (field programmable gate array) or an ASIC(application-specific integrated circuit).

Processors suitable for the execution of a computer program include, byway of example, both general and special purpose microprocessors, andany one or more processors of any kind of digital computer. Generally, aprocessor will receive instructions and data from a read-only memory ora random access memory or both. Elements of a computer may include atleast one processor for executing instructions and one or more memorydevices for storing instructions and data. Generally, a computer alsomay include, or be operatively coupled to receive data from or transferdata to, or both, one or more mass storage devices for storing data, forexample, magnetic, magneto-optical disks, or optical disks. Informationcarriers suitable for embodying computer program instructions and datainclude all forms of non-volatile memory, including by way of examplesemiconductor memory devices, for example, EPROM, EEPROM, and flashmemory devices; magnetic disks, for example, internal hard disks orremovable disks; magneto-optical disks; and CD-ROM and DVD-ROM disks.The processor and the memory may be supplemented by, or incorporated inspecial purpose logic circuitry.

To provide for interaction with a user, implementations may beimplemented on a computer having a display device, for example, acathode ray tube (CRT) or liquid crystal display (LCD) monitor, fordisplaying information to the user and a keyboard and a pointing device,for example, a mouse or a trackball, by which the user can provide inputto the computer. Other kinds of devices can be used to provide forinteraction with a user as well; for example, feedback provided to theuser can be any form of sensory feedback, for example, visual feedback,auditory feedback, or tactile feedback; and input from the user can bereceived in any form, including acoustic, speech, or tactile input.

Implementations may be implemented in a computing system that includes aback-end component, for example, as a data server, or that includes amiddleware component, for example, an application server, or thatincludes a front-end component, for example, a client computer having agraphical user interface or a Web browser through which a user caninteract with an implementation, or any combination of such back-end,middleware, or front-end components. Components may be interconnected byany form or medium of digital data communication, for example, acommunication network. Examples of communication networks include alocal area network (LAN) and a wide area network (WAN), for example, theInternet.

Example 1

In one embodiment of the device to determine the identity of apathogenic bacteria in blood, the microfluidic separation stage is asdescribed in FIG. 3 . Whole blood is diluted using water about ten-foldand enters the microfluidic chip. The amount of blood cells (white bloodcells, red blood cells, and platelets) is reduced using the blood cellreducer using the inertial forces generated by the spiral flow path.While most of the blood cells are eliminated as a waste stream, theintact bacteria, some blood cells, other non-bacterial cells of similarsize to the intact bacteria, and plasma move to the next sub-stage—theporous polymer monoliths. The blood cells entering this stage and othernon-bacterial cells are selectively lysed taking advantage of theirhigher sensitivity to mechanical stresses yielding a fluid stream thatconsists of intact bacteria, lysis debris, and plasma. This fluid streammoves to the DLD sub-stage where the intact bacteria are separated fromthe rest of the components on the basis of the difference in theirhydrodynamic size. The volume of the fluid containing the intactbacteria is reduced by about 10-10000-fold during this sub-stage. Thissmall volume of fluid containing intact bacteria and plasma proteinsnext moves to the nanofilter. The plasma proteins pass through the poresof the filter along with water and other small molecules (for example,glucose, cholesterol, and the like) leaving the intact bacteria on thesurface of this nanofilter. The surface area of the filter is about0.01-0.3 mm². Processing a 1 mL sample volume at 10 CFU/mL in the mannerdescribed above leads to an effective concentration of 10⁶ CFU/mL attime of measurement assuming 100% recovery of intact bacteria,measurement area of 0.01 mm², and a film thickness of 1 micron.

By processing a relatively large volume (1 mL) of blood, the probability(and the amount of) bacterial presence in the sample is increased. Sincebacteria are not distributed homogeneously throughout the clinicalsample, a 0.1 mL sample of blood is not assured of containing 1 CFU ofbacteria. The concentration of the intact bacteria enablesidentification without requiring culturing and isolation.

The infrared spectrum is collected using an FTIR spectrometer such asthe Vertex 80 ® from Bruker equipped with a microscope accessory such asthe Hyperion® (Bruker). This is typically performed at about a 4 cm⁻¹resolution. The background spectrum is first collected using nanofilterbefore bacterial concentration as the background. The spectrum of thebacteria on the nanofilter surface (sample spectrum) is acquired. Thebackground spectrum and atmospheric contributions (such as those due tocarbon dioxide and water vapor) are then subtracted from this samplespectrum. The second derivative of the resulting spectrum is thenacquired to determine the position of the peaks over the range fromabout 400 to 4000 cm⁻¹. The position of the peaks is represented by thelocal minima of the second derivative of the spectrum. These positionsare compared against those in the reference database. In addition, thematrix of the sample spectrum (after subtraction of the background andatmospheric contributions) consists of the wavenumber and intensity ofabsorbance at each wavenumber. These spectral profiles are also furthersmoothed using a 9-point Savitzky-Golay algorithm.

Cluster analysis is conducted according to Ward's algorithm [36]. Inthis method, the criterion for choosing the pair of clusters to merge ateach step is based on the optimal value of an objective function, suchas the error of the sum of squares. At each step of the analysis,clusters of data with the minimum inter-cluster distance are merged.This assessment is used to classify the different pathogens and theresulting method was stored in a database. The sample spectrum isanalyzed against this database to determine which of the pathogenicspectra it most closely resembles. This is reported as the identity ofthe bacteria.

Example 2

The presence and identification of bacteria in apharmaceutical-containing solution such as a monoclonal antibodybiopharmaceutical preparation is determined by the detection system asgenerally described above. The microfluidic separation stage resemblesthe one shown in FIG. 3 . However, the blood cell reducer sub-stage isremoved from the microfluidic chip. Very few non-bacterial cells areexpected in such a sample. Despite this, the sample stream is made toflow through the lysis stage. Any non-bacterial cells present would beselectively lysed yielding intact bacteria, lysis debris, andpharmaceutical-containing solution.

This fluid stream moves to the lysis-debris remover sub-stage where theintact bacteria are separated from the rest of the components on thebasis of the difference in their hydrodynamic size. Most of thepharmaceutical product is separated from the intact bacteria along withany lysis debris. The volume of the fluid containing the intact bacteriais reduced by about 10-10000-fold during this sub-stage. This smallvolume of fluid containing intact bacteria moves to the nanofilter. Anyresidual component (for instance, formulation components such asbuffering agent) is filtered, leaving the intact bacteria on the surfaceof this nanofilter. The bacteria on the surface of this nanofilter areidentified as described in Example 1.

Example 3

Bacterial contamination of milk is ascertained by the detection system.In this case, milk is diluted using water about 10-fold and enters themicrofluidic chip. The concentration of the proteins, fats, and othercomponents of milk is reduced by dilution and by passage through aspiral path. This configuration is the similar to the one shown in FIG.3 . Inertial forces are used to reduce the level of components smallerthan the bacteria; the bacteria-containing stream is directed towardsthe lysis sub-stage while the rest of the stream is directed to a wastereceptacle. The lysis sub-stage is used to lyse any non-bacterial cells(for instance, epithelial cells from a cow) leaving intact bacteria topass onto the lysis debris removal stage. In this embodiment, the lysisdebris removal stage is also a spiral flow path that separates the lysisdebris from bacteria on the basis of their hydrodynamic size. The intactbacteria-containing solution is collected on the surface of anIR-compatible surface, such as zinc selenide, and allowed to dry. Thesurface of the zinc selenide is then mapped using an array detector.Areas, containing distinctive bacterial absorbance patterns areidentified and the bacterial signature compared against the referencedatabase as described before. If no bacterial absorbance patterns areidentified, the absence of bacteria in the sample is reported as theresult.

The invention described in this specification generally relates todevices and associated methods of detecting and identifyingmicroorganisms such as bacteria and other small and very smallmulticellular microorganisms. While certain exemplary embodiments havebeen described above in detail and shown in the accompanying drawingfigures, it is to be understood that such embodiments and examples aremerely illustrative of and not restrictive of the broad invention. Inparticular, it should be recognized that the teachings of the inventionapply to variations of the preferred embodiments that are specificallydiscussed.

While certain features of the described implementations have beenillustrated as described herein, many modifications, substitutions,changes and equivalents will now occur to those skilled in the art.Thus, it will be understood that the invention is not limited to theparticular embodiments or arrangements disclosed, but is rather intendedto cover any changes, adaptations or modifications which are within thescope and spirit of the invention as defined by the appended claims.

REFERENCES

All of the journal articles and all other publications, patents andtexts mentioned in this specification are incorporated by reference intheir entireties, including the following:

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The invention claimed is:
 1. A detection system for identifyingmicroorganisms within a sample, the sample comprising a fluid andincluding non-microbial cells and microorganisms, the detection systemcomprising: a microfluidic separation stage comprising a lysis structurethat includes an inlet, an outlet, and microporous surfaces disposedbetween the inlet and the outlet to receive the sample and selectivelylyse non-microbial cells while maintaining intact microorganisms in thesample when the sample flows between the inlet and the outlet andthrough the microporous surfaces of the lysis structure; a detectionstage to receive the sample from the microfluidic separation stage andperform detection analysis of microorganisms in the sample.
 2. Thedetection system of claim 1, wherein the microorganisms are selectedfrom the group consisting of bacteria, fungi, and small unicellular andmulticellular organisms.
 3. The detection system of claim 1, wherein thesample is a liquid selected from the group consisting of whole blood,plasma, serum, saliva, urine, cerebrospinal fluid, water, and fruit andvegetable juices.
 4. The detection system of claim 1, wherein the sampleis selected from the group consisting of a fluid derived from meat,produce, processed food, dairy products, poultry products,pharmaceutical process streams, bulk drug substance, and final drugproduct.
 5. The detection system of claim 1, wherein the volume of thesample ranges from about 0.1 mL to about 10 mL.
 6. The detection systemof claim 1, wherein the microfluidic separation stage further comprises:a first separation stage to separate components that are larger in sizethan the microorganisms for detection, wherein the lysis structurereceives the sample from the first separation stage at the inlet of thelysis structure; and a second separation stage to receive the samplefrom the outlet of the lysis structure, the second separation stage toseparate lysed cells and cellular debris from the microorganisms fordetection.
 7. The detection system of claim 1, wherein the microfluidicseparation stage concentrates the microorganisms in the sample by aprocess selected from the group consisting of reduction in total samplevolume and filtration.
 8. The detection system of claim 1, furthercomprising: an identification stage to identify intact microorganismsbased upon the detection analysis.
 9. The detection system of claim 8,wherein the identification stage further comprises a measurement stagethat measures the signatures of the microorganisms in transmission mode.10. The detection system of claim 9, wherein the measurement stagefurther comprises a flow cell or detection window.
 11. The detectionsystem of claim 9, wherein the measurement stage comprisespolycrystalline silicon.
 12. The detection system of claim 9, whereinthe identification stage further comprises a computer system.
 13. Thedetection system of claim 9, wherein the identification stage furthercomprises a reporting stage.
 14. The detection system of claim 1,further comprising a waste collection stage.
 15. The detection system ofclaim 1, further comprising a control stage.
 16. The detection system ofclaim 1, wherein the microorganisms are selected from the group ofgenera consisting of Staphylococcus, Escherichia, Listeria, Salmonella,Streptococcus, Klebsiella and Campylobacter.
 17. The detection system ofclaim 1, wherein the detection system is portable.
 18. The detectionsystem of claim 1, wherein the microporous surfaces are sized to permitpassage of intact microorganisms in the sample through the microporoussurfaces while lysing non-microbial cells.
 19. The detection system ofclaim 1, wherein the microporous surfaces include pores having diametersfrom 1 micrometer to 10 micrometers.
 20. The detection system of claim1, wherein the lysis structure comprises at least one porous monolithicstructure disposed between the inlet and the outlet of the microfluidicseparation stage, and the porous monolithic structure includes themicroporous surfaces.